FDA-approved Abl/EGFR/PDGFR kinase inhibitors show potent efficacy against pandemic and seasonal influenza A virus infections of human lung explants

Summary Influenza viruses (IVs) cause substantial global morbidity and mortality. Given the limited range of licensed antiviral drugs and their reduced efficacy due to resistance mutations, repurposing FDA-approved kinase inhibitors as fast-tracked host-targeted antivirals is an attractive strategy. We identified six FDA-approved non-receptor tyrosine kinase-inhibitors (NRTKIs) as potent inhibitors of viral replication of pandemic and seasonal IVs in vitro. We validated their efficacy in a biologically and clinically relevant ex vivo model of human precision-cut lung slices. We identified steps of the virus infection cycle affected by these inhibitors and assessed their effect(s) on host responses. Their overlapping targets suggest crosstalk between Abl, EGFR, and PDGFR pathways during IAV infection. Our data and established safety profiles of these NRTKIs provide compelling evidence for further clinical investigations and repurposing as host-targeted influenza antivirals. Moreover, these NRTKIs have broad-spectrum antiviral potential given that their kinase/pathway targets are critical for the replication of many viruses.


INTRODUCTION
Influenza viruses (IVs) cause respiratory tract infections in humans and are responsible for substantial annual morbidity and mortality, especially in individuals at high risk, like young children, the elderly, or immunocompromised patients. The most important preventative measure of protection from IV infections is vaccination. However, accumulation of genomic mutations contributes to reduced vaccine efficacy and evasion of virus-neutralizing antibodies; necessitating annual update vaccines against seasonal IVs. [1][2][3][4] Moreover, genetic reassortment that leads to the emergence of novel IVs in human populations, that largely lack virus-neutralizing antibodies, can result in pandemic outbreaks. As with previous influenza pandemics and the current SARS-CoV-2 pandemic, effective vaccines are not readily available at early stages of a pandemic.
In absence of efficacious vaccines, virus-targeted antivirals offer some protection if administered within the therapeutic window. Until recently, only two classes of influenza antivirals were available, adamantanes targeting the viral M2 ion channel protein and neuraminidase inhibitors (NAI). However, adamantanes are ineffective due to resistance mutations in currently circulating strains and are no longer clinically used. 5 In contrast, only $4-5% of currently circulating viruses carry resistance mutations to NAIs (like oseltamivir). 6 Favipiravir (T705), baloxavir and pimodivir are recently developed antivirals targeting the viral PA, PB1, and PB2 polymerase proteins, respectively, and can inhibit adamantane-and NAI-resistant viruses. 7,8 Although baloxavir was approved for the treatment of acute ''uncomplicated'' influenza infections in the United States in 2018, recent studies have already observed $10% of isolates from otherwise healthy adults and adolescents carried baloxavir resistance mutations; this may potentially be higher in immunocompromised patients. [9][10][11][12] Sustained circulation of virus variants resistant to current antivirals and low genetic barrier for resistance highlight the need for host-targeted therapeutics; that do not suffer from these limitations. Because all viruses rely on cellular machinery for their replication, several host proteins are known to be required for efficient viral replication and pathogenesis. [13][14][15][16][17] Host kinases regulate signaling pathways that are critical for

Validation of NRTKIs as IAV antivirals in hPCLS
We utilized human precision-cut lung slices (hPCLS) as a biologically relevant ex vivo model that more faithfully represents lung tissues than either 2D monolayer or 3D air-liquid-interface (ALI) cultures. 33,34 We used hPCLS cultured up to 4 weeks; no gross alterations in cell type or morphology were observed and cilial beating was observed. We infected hPCLS with 10 4 , 10 5 , or 10 6 TCID 50 /200 mL of NL09 or NL11 to identify an infectious dose where peak titers of NL09 or NL11 are synchronized; these strains have different replication kinetics in vitro. To limit donor-heterogeneity effects, we used hPCLS from 8 donors (n = 24/virus). Highest peak titers were achieved at 48 hpi following infection with either 10 4 or 10 5 doses of NL11, but only in the 10 5 dose of NL09 ( Figure 3A); therefore, the 10 5 dose was used in all subsequent hPCLS infections.
Next, we determined the tolerability of our hPCLS to either [1x or 10x] max NRTKI concentrations by measuring lactate dehydrogenase (LDH) release into the culture supernatant as an indicator for cytoxicity. As a positive control for cytotoxicity, hPCLS were treated with 0.1% Triton X-100; DMSO-treated hPCLS were used as a vehicle control ( Figure 3B). Our cytotoxicity cut-off was 20% of the positive control treatment; none of the NRTKIs surpassed this cut-off at [1x] max . However, 10x max concentrations of DF (50 mM), BO (50 mM), and SA (1.25 mM) showed significantly higher cytotoxicity (>20%); we therefore, only used 1x concentrations of these NRTKIs in subsequent hPCLS experiments ( Figure 3B).
Next, hPCLS from 3 donors (n = 6/virus/condition) were infected with 10 5 TCID 50 of NL09 or NL11 and treated with NRTKIs (DF 5 mM; AC 5 mM; IB 5 mM; BO 5 mM; NI 10 mM; SA 0.125 mM). All tested NRTKIs reduced viral titers by at least 10-fold or 1-log 10 (DF treatment) to more than 1,000-fold or 3-log 10 (IB and NI treatments) ( Figure 3C). However, while this effect was significant throughout the infections with either NL09 or NL11 following IB, BO, and NI treatments, AC and DF treatment was only significant at 24 and 48 hpi. Moreover, unlike what we observed in A549 cells, IB-, BO-, and NI-mediated IAV inhibition was observed within 12 hpi and maintained at 120 hpi; which was after the times of peak titers (48-72 hpi) ( Figure 3C).
Next, we assessed NRTKI effects on the viral spread and associated damage to the epithelium. At 120 hpi, mock-and IAV-infected hPCLS (n = 3/virus/condition) were fixed and paraffin-embedded. H&E staining did not suggest gross alterations in cell composition or epithelium in mock-infected cells indicating acceptable viability of hPCLS; typical morphological changes associated with IAV infections were observed. Only data for NL11 is shown as we did not observe significant differences in titer reductions between NL09 and NL11 ( Figure 3D). We observed IAV-specific staining in all observed cell types including type I/II pneumocytes and endothelial cells in consecutive sections from those H&E stained. Additionally, staining intensity and quantity were reduced in NRTKI-treated hPCLS compared to untreated hPCLS ( Figure 3D). Semi-quantitative analysis of acquired images indicated that DF, AC, BO, and SA treatment significantly reduced infectivity by >50%, whereas IB and NI treatment had limited effects on infectivity ( Figure 3E).

Stability of NRTKI inhibition
Host-directed antivirals/therapeutics likely have a higher barrier of resistance than their virus-targeted counterparts. To determine the stability of NRTKI antiviral effect, we phenotypically assessed the emergence of resistant escape variants. 35,36 We passaged both NL09 and NL11 viruses (MOI = 0.001) in the presence of NRTKIs [1x] max in MDCK cells for 5 passages. Untreated virus stocks were also passaged as a control. In untreated passages, virus titers for both NL09 and NL11 were similar from passages 1 to 5 and were significantly lower in all treated passages ( Figure 4A) iScience Article A549 cells. Viral titers were stable at all passages indicating no resistance mutations were acquired. Next, we ruled out NRTKI virucidal activity or direct NRTKI-virus interactions that may inhibit attachment or entry. Pre-treatment of virus stocks with NRTKIs [1x] max for 2 h prior to A549 infection had no effect on viral titers, indicating that the observed effects are due to host-cell effects ( Figure 4B).  Kinases regulate every step of the infection cycle and a single kinase can affect multiple steps. 28,29 To determine the effect of our NRTKIs on viral entry, A549 cells were pretreated for 2 h, then infected with NL09 or NL11 (MOI = 10). At 0.5 hpi, cells were fixed, stained to detect viral NP, F-actin, and nuclei, and analyzed by confocal microscopy. We observed significant retention at the membrane and cell periphery following DF and IB treatment ( Figure 5). Surprisingly, no virus was detected in response to BO treatment and the F-actin network was not detectable. Given the sustained viability of BO-treated cells, it is likely that the altered actin dynamics were tolerated. No significant changes were detectable in AC-, NI-, or SA-treated cells suggesting they did not affect viral entry ( Figure 5).

NRTKIs exert differential effects on IAV polymerase activity
We next assessed the effect of our NRTKIs on viral polymerase activity. The pPOLI-358-FFLuc reporter plasmid, which encodes a firefly luciferase gene under the control of the viral nucleoprotein (NP) promoter, and luciferase activity is a surrogate for viral polymerase activity. [37][38][39] We first compared the effect of NRTKIs on polymerase activity during infection. We observed a significant reduction in polymerase reporter activity in response to AC (NL11 only), IB (NL09 only), BO, NI, and SA (NL11 only) ( Figure 6A). Although the magnitude of reduction was higher in NL09-infected than NL11-infected cells, a significant A B Figure 4. NRTKI inhibition of IAV is stable (A) Stability of SMKI treatment on NL09 and NL11 replication was determined by serial passaging (5 times iScience Article reduction was more readily observed in NL11-infected cells at lower NRTKI concentrations. DF reduced reporter activity (NL09 = 20%, NL11 = 13%); however, this reduction was not statistically significant. At 24 hpi, a 3-fold increase in reporter activity could be observed in untreated NL11-infected cells over untreated NL09-infected cells; this is in line with faster replication kinetics of NL11 compared to NL09 ( Figure 6B).
To better dissect the direct effect on viral polymerase activity in absence of NRTKI-effects on viral entry and host responses, we determined polymerase activity using an established minigenome system that only expresses the viral replication complex proteins (NP, PA, PB1, and PB2). We used the minigenome from the related H3N2 strain NL03 as we did not have access to the NL11 minigenome plasmids; both strains are known to exhibit similar replication kinetics. In this context, AC (NL09 only), IB, NI, and SA treatments significantly reduced polymerase activity ( Figure 6C). In contrast to infected cells, the magnitude of reduction in polymerase activity was comparable in NL09-and NL03-minigenome transfected cells. Interestingly, polymerase activity was significantly higher in untreated H1N1 (NL09) than in H3N2 (NL03) minigenome-transfected cells ( Figure 6D).

NRTKIs do not affect innate immune responses during IAV infections
STAT3 is a regulator of inflammatory responses that is activated by phosphorylation of Y705 (STAT3pY705) to upregulate anti-apoptotic factors. This activation is less efficient in H1N1 infections than in H5N1 infections which delays apoptosis more efficiently. Therefore, we determined if NRTKI-mediated inhibition of viral entry and/or replication affected STAT3pY705. We assessed STAT3pY705 in A549 cells infected with either NL09 or NL11 (MOI = 1) in the presence or absence of NRTKIs at [1x] max . As expected, decreased . Surprisingly, only DF treatment resulted in a significant reduction of pSTAT3 relative to untreated infected cells (NL09 = 5%-8%, NL11 = 5%-14% of untreated).
NFkB Activation (NFkBpS536) serves opposing functions in early vs later times of infection and is therefore tightly regulated during IAV infection. We did not detect a significant increase in relative NFkBpS536 in NL09-or NL11-infected cells compared to mock-infected cells at 18 or 48 hpi following treatment with any of the NRTKIs ( Figure 7B). We did observe a reduction in NFkB activation following DF treatment of NL11-infected cells (18 h = 64%, 48 h = 68% of untreated); however, this reduction was not statistically significant. We also confirmed that NFkB signaling is not impaired in our system as treatment with the synthetic dsRNA activator, poly(IC), induced NFkBpS536 in mock-, NL09-,and NL11-infected cells at 18 and 48 hpi ( Figure S3). iScience Article FDA-approved NRTKIs currently in clinical use against cancers and autoimmune diseases for their antiviral potential against IAV infections. Six of eight tested NRTKIs showed potent in vitro inhibition of pandemic (H1N1) and seasonal (H3N2) IAV strains with little to no impact on cell viability in vitro. The robust potency was further validated and confirmed for five of six NRTKIs using a faithful ex vivo model of human PCLS. We identified the step(s) of the viral replication cycle affected by each compound. In doing so, we provide valuable information on the interplay of signaling pathways regulating these steps and the likely kinases involved.
We initially used A549 cells (ATII lung adenocarcinoma); however, due to potential biases associated with aberrant expression and kinase activity of cancerous cell lines, we validated NRTKI candidates using human PCLS (hPCLS) from 11 donors in total. Unlike 2D monolayers or 3D well-differentiated air-liquid interface (ALI) cultures, PCLS preserve native lung tissue architecture, cellular composition including endothelial, ATI and ATII epithelial cells, fibroblasts, and maintain native extracellular matrix. 33,34,40 Moreover, tissue tropism and infectivity of certain viruses may not be accurately represented in vitro due to the absence of relevant cell-cell interactions that influence infectibility and host responses. 41 Accordingly, we observed a similar discrepancy in which strain-dependent variances observed in NRTKI-treated A549 cells were not observed in hPCLS, suggesting that the differences between IAV strains in A549 might be an in vitro artifact. Moreover, we observed wide tissue tropism in hPCLS which suggests that while ATII cells may support more efficient infection, other cell types of the lung are readily infectible as well. Nevertheless, robust viral titer reductions in both systems following NRTKI treatment were observed that were not readily explained by reduction in infectivity or cell viability only. Defactinib (DF) < Acalabrutinib (AC) < Saracatinib (SA) < Bosutinib (BO) treatment had the smallest effect on viral titers, but each of these NRTKIs reduced hPCLS infectivity by $50%. Similarly, Ibrutinib (IB) and Nilotinib (NI) had the largest effect on viral titers and actually increased infectivity (NI:$22%, IB = $7%). Together our data indicate that the reduction in infectivity of either A549 cells or hPCLS does not fully account for the potent reduction in viral titers. The rate at which viruses infect cells can indeed influence viral titers. However, the correlation between infectivity and titers is not absolute, and may only be readily observed when using a compound that targets the virus itself. Infectivity involves viral entry and replication at the very least, and in our assays, we carried out the infections at an MOI of 1 to allow for effects on virus spread. While the same number of cells may be infected, compound-specific effects on viral entry, polymerase activity, and/or virion assembly/egress could vastly differ and result in an uncoupling of the virus infectivity-titer correlation. Therefore, we do not believe that our findings regarding infectivity/viral titers are mutually exclusive or contradictory and support a bona fide effect of NRTKIs on viral entry and/or RNA replication.
Host kinases play a critical role in IAV entry, replication, and release as well as viral evasion/suppression of hosts' immune responses; processes often requiring phosphorylation of viral proteins by mostly still unidentified kinases. [42][43][44][45][46][47][48][49][50][51][52][53] Although there is a growing body of in vitro and in vivo evidence to support the therapeutic potential of kinase inhibitors, not a single SMKI has been approved or licensed for the treatment of influenza virus infection so far. 1,13,14,17,48 SMKI selectivity remains a contentious topic and has been a hurdle to the pursuit of kinase inhibitors as antivirals. While compounds still in pre-clinical development phases require target validation, the selectivity of compounds in clinical use has been heavily investigated. 21,[54][55][56] Phosphorylation or activation of proteins/pathways beyond the intended target are often regarded as ''offtarget effects''. However, kinase-substrate interactions highlight extensive crosstalk between signaling pathways. Therefore, inhibition of an ''off-target'' kinase or pathway, should not be oversimplified and attributed to promiscuity of the SMKI in question; rather it is more likely evidence of an interaction between the intended target and the affected ''off-target'' signaling node. Two seminal studies collectively examined the selectivity of over 170 SMKIs against more than 440 kinases, covering $80% of the human kinome. Davis et al. compared inhibitor-kinase binding affinities, whereas Anastassiadis et al. used functional kinase inhibition assays. These studies suggest that while classes of SMKIs can inhibit multiple kinases within a single subfamily, inhibitors are selective against kinases outside that subfamily. 55,56 Moreover, these studies likely overestimated ''off-target'' effects due to the use of truncated or fused recombinant proteins that may adopt altered conformations in the absence of regulatory domains (i.e., regulatory subunit of PI3K) or binding partners that affect substrate or ATP binding sites availability 21 ; suggesting even greater selectivity than was proposed by those studies. Likewise, selectivity of clinically approved SMKIs was validated by super-resolution microscopy (dSTORM) to show the superior specificity and selectivity of fluorescently labeled SMKIs like gefitinib (EGFR inhibitor), over either fluorescent EGF ligand or EGFR monoclonal antibody. 57 Therefore, SMKIs can be used as molecular beacons to probe host signaling pathways and delineate how they are regulated by kinases. iScience Article NRTKIs which target known effectors of actin reorganization and endocytosis have a significant effect on IAV entry. Indeed, we previously showed that targeting FAK using the pre-clinical inhibitor Y15, led to inhibition of PI3K-mediated endosomal trafficking of virions. 28 Using the FDA-approved FAK inhibitor DF, we saw comparable effects on actin reorganization and viral entry as we previously observed using Y15. 28 This is consistent with DF having the most effect at earlier time points in both A549 and hPCLS when reduction in viral entry may have a larger impact than at later time points. Indeed, we observed a reduction in infectivity in response to DF treatment which is consistent with the observed reduction of viral entry.
Cell-specific Bruton's tyrosine kinase (BTK) isoforms are implicated in PI3K and PLCg signaling that are either pro-or anti-apoptotic. 58 IB and AC are high-affinity irreversible inhibitors of BTK; IB also inhibits EGFR activity. 59,60 IB treatment reduces excessive neutrophil infiltration, acute lung injury, and subsequent ARDS; ultimately resulting in increased survival of mice severely infected with IAV. 27 Given that IB inhibits both EGFR and BTK whereas AC selectively inhibits BTK, the IB-specific reduction in viral entry we observed, suggests this effect is mediated largely through inhibition of EGFR signaling. This is consistent with IAV-induced EGFR signaling which facilitates viral entry and activation of downstream pathways (Src, PI3K, and ERK) that promote efficient replication. 61 BO, along with NI and SA, are second-generation Src inhibitors. BO also inhibits Abl kinase and to a lesser extent BTK. Growth factor RTKs like PDGFR and EGFR induce Src-mediated activation of PI3K/AKT, Ras-Raf-MEK-ERK, FAK, and STAT3. 62 Src's mostly proviral role during IAV infections is modulated by the viral NS1 protein. 13,63 Abl inhibition by some avian IAVs results in significant pathology in vitro and in vivo; however, Abl's role in human IAV infections is not fully understood. 64, 65 The direct interactions of a specific avian NS1 motif with Abl are necessary for the disruption of Abl kinase activity and result in significant cytopathic effect. 64,65 Interestingly, this motif was present in the pandemic 1918 IAV strain when it first crossed into humans but was quickly lost during human adaptation so recent circulating human IAVs do not carry this motif suggesting that Abl kinase activity is important for human adaptation of IAVs. In the context of cellular functions, Abl is activated by both Src-dependent and -independent EGFR and PDGFR signaling. Among its functions is cytoskeletal reorganization which is at least partially mediated through Src signaling. Interestingly, Abl activity seems to positively regulate EGFR receptor endocytosis 66 ; establishing a possible link for Abl to EGFR-mediated IAV entry. This is consistent with the significant reduction in viral titers we observed following BO treatment of hPCLS and A549 cells. BO treatment also resulted in a stark reduction in viral entry, concurrent to a complete absence of detectible actin filaments despite an increase in cell viability during infection. This is consistent with reports that show BO inhibition of Src activity can lead to altered actin dynamics or enhanced depolymerization of F-actin due to retention of alpha-and b-catenin at the cell membrane 67,68 ; a process that may be influenced by Abl activity which is also affected by BO treatment.
We used a polymerase activity reporter and mini-genome systems to better dissected the effect of our NRTKIs on RNA replication and polymerase activity. 39 Interestingly, in the context of viral infections, we detected higher polymerase reporter activity in NL11 (H3N2)-infected cells than in NL09 (H1N1)-infected cells. In contrast, the opposite was true when using the minigenome. Faster replication kinetics may be more susceptible to NRTKIs as a reduction in replication rate leads to exponential differences with time. This suggests that polymerase activity of NL09 is higher than NL11 and the faster kinetics in virus replication observed in NL11 infected cells may be due to more efficient virus entry, release, or immune evasion than NL09, and not polymerase activity. We previously demonstrated that FAK, in addition to its role in viral entry, also regulates in vitro polymerase activity of multiple IAV strains using the selective FAK inhibitor Y15 or dominant-negative kinase mutants. 29 However, in contrast to our previous studies, we only observed a modest and non-significant effect on polymerase activity following DF treatment. O'Brien et al. showed that Y15 was a significantly more potent and selective inhibitor of FAK activity than DF which also targets the FAK-related kinase Pyk2. 69 The disparity between a given SMKI's binding affinity (K d ) and its functional inhibitory concentrations can also be observed in the case of a single inhibitor targeting multiple kinases. For instance, the K d of the multi-kinase inhibitor sunitinib for TrkC is 5.1 mM, but a 10-fold lower concentration (0.5 mM) is sufficient to inhibit >97% of its activity. In contrast, sunitinib's K d for PAK3 is 16 nM, but not even a 30-fold higher concentration (0.48 mM) has an effect on its activity. 54  iScience Article not directly affect IAV RNA replication. In contrast, inhibition of Abl and PDGFRa by NI treatment had the most significant reduction in IAV polymerase activity that was also strain independent. These data point to a role of PDGFRa in facilitating efficient IAV polymerase activity. This is consistent with previous findings that show inhibition of PDGFR by the RTK inhibitor A9, blocks RNA synthesis of all viral RNA species (vRNA, cRNA, and mRNA) independently of NFkB signaling. 44 However, A9 also inhibits EGFR and is not selective for PDGFR isoforms; whereas NI is >25-fold more selective for PDGFRa than PDGFRb. 70 Several reports indicate that IAVs modulate antiviral NFkB activity to facilitate viral replication. Inhibition of NFkB results in reduced viral titers partly due to a disruption of vRNP nuclear export. [71][72][73] Although we observed induction of NFkB activation by poly(IC) treatment in mock-and IAV-infected cells at 18 h but not 48 h, we did not observe a robust induction in NFkB phosphorylation in IAV-infected cells without poly(IC) treatment. This is consistent with published data and most likely due to the immuno-suppressive role of the viral NS1 protein. 74 FAK also modulates cellular immune responses by regulating T cell-, B cell-, and macrophage-functions as well as RIG-I-Like antiviral signaling. [75][76][77][78] We previously demonstrated FAK-dependent regulation of NFkB signaling and polymerase activity in vitro and NFkB-dependent proinflammatory responses in vivo. 30 In that study, FAK inhibition increased survival, reduced viral load and pathogenesis in a severe infection model. However, DF treatment did not affect NFkB phosphorylation in this study; likely due to the difference in FAK inhibition potency between Y15 and DF. Similarly, none of the other NRTKIs influenced NFkB activation suggesting that the mechanism by which these NRTKIs inhibit virus replication is independent of the NFkB-pathway. Considering the transient and biphasic nature of NFkB activation, we cannot rule out that strain-dependent differences in kinetics did not affect the magnitude or duration of NFkB activation we observed as has previously been described by others. [79][80][81][82][83][84] STAT3 is an emerging regulator of IFN and inflammatory responses. A wide range of cytokine, growth factors, and RTKs activate STAT3 via JAK1/2/3 and Tyk2-dependent phosphorylation at Y705 (STAT3pY705). 85 The role of STAT3 is not fully understood with opposing functions dependent on pathway partners; IL-6 mediated STAT3 activation is proinflammatory while IL-10 mediated STAT3 activation is anti-inflammatory. 85,86 Although STAT3 is dispensable for IFN signaling, it is activated by IFN-I and serves as a negative regulator to fine-tune the IFN response (reviewed in 86 ). Recent studies suggest that STAT3 activation is likely IAV subtype/strain-dependent as well as host/tissue-specific. [87][88][89] Because STAT3 activation upregulates anti-apoptotic factors, H5N1-mediated STAT3pY705 allows prolonged viral production through a delay of apoptosis; H1N1 is less efficient at STAT3pY705 and triggers apoptosis earlier. 88,89 Although the mechanism of differential suppression of STAT3 activation by IAV is not clear, it has been suggested to be mediated by NS1 74 . Interestingly, EGFR activation can result in Src/FAK/BTK mediated activation of STAT3, thereby modulating the IFN and proinflammatory responses. Moreover, EGFR/Src-mediated STAT3pY705 requires Pyk2 kinase activity, which can also mediate full STAT3 transcriptional activity via JNK, p38, or ERK activation. 90 As expected of H1N1 and H3N2 infections, 88,89 we observed limited levels of STAT3pY705 in untreated cells that were comparable to that observed following treatment with most NRTKIs. However, we observed significant suppression of STATpY705 following DF treatment (7-14% of untreated infected cells). This is consistent with the fact that DF inhibits both FAK and Pyk2 and suggests that IAV-induced STAT3pY705 requires FAK and/or Pyk2 activity.
In summary, we demonstrate that NRTKIs target kinases required for efficient IAV replication and represent promising drugs for the development of the next generation of antivirals. It is tempting to speculate on the molecular mechanisms and contribution of individual kinases to the antiviral effects observed following NRTKI treatment. The NRTKIs with the greatest effect had overlapping targets that mainly included EGFR, PDGFRa, and Abl. In contrast, NRTKIs that mainly target BTK or Src or the more distant FAK/ PyK2 family had less robust, but still biologically significant, effects on viral replication. This data could be further used to fine-tune selectivity and potency for next-generation antiviral SMKIs and provide a rationale for combination therapy options to maximize SMKI antiviral efficacy. Surprisingly, our tested NRTKIs directly affected steps of the virus replication cycle with limited effects on proinflammatory host responses. Nicholas  iScience Article often smokers and suffer from either COPD or other respiratory pathologies. Although at first glance this may be perceived as a limitation of our model, we believe that these donors represent the ''at risk'' populations that would most benefit from IAV antivirals. Therefore, our data obtained from donor PCLS using these already FDA-approved NRTKIs as IAV antivirals are highly applicable to clinical settings. It should be noted however, that because these inhibitors target host factors, their therapeutic window is likely to be different from that of virus-targeted antivirals and pre/clinical studies must take this into account to establish efficacy.
In contrast to virus-directed IAV antivirals which are susceptible to resistance mutations, our tested NRTKIs data have a high genetic barrier for resistance based on their stability of IAV inhibition after 5 passages in the presence of each of our six NRTKIs. Although we cannot rule out NRTKIs-selected mutations, we did not detect a significant change in the magnitude of viral titer reductions across 5 passages, suggesting that no mutations conferring resistance accumulated in viruses passaged in the presence of SMKIs. Additionally, their established safety and bioavailability data further warrants clinical evaluation of these compounds as potential influenza treatments. Given that IAV infections are typically restricted to the respiratory tract, localized delivery of kinase inhibitors can limit potential cytotoxic effects. Finally, the local microenvironment must be considered to elicit balanced immune responses and avoid opposite or unintended consequences of promising SMKIs on resident and infiltrating immune cells. Because many viruses utilize the same (or related) host kinases to facilitate replication and transmission, our studies have broader implications for the potential use of these SMKIs to treat infections by other viruses.

Limitations of study
Due to the dependence on available hPCLS, human lung samples of older (>58 years old) mainly male patients (10 male/1 female) undergoing tumor resection (8 tumor resections/3 IPF) were overrepresented. Also, A549 cells were originally derived from a 58 years old male patient with lung cancer. Although the female and the IPF patient samples showed no differences in their susceptibility or response to the NRTKI treatment, we cannot exclude potential bias of our data based on sex, medical condition, or age. Since older adults are at high risk for influenza, our findings are particularly relevant for this age group. IAV has multiple mechanisms to actively suppress p65 phosphorylation at specific times of infection. However, it is apparent that p65 phosphorylation in IAV-infected cells is not straightforward and a matter of debate. Differences in experimental conditions like strains used, multiplicities of infection and kinetics, as well as methods of measuring p65 activation (e.g., p65 phosphorylation only, phosphor/total p65 ratio, p65 nuclear translocation), could be at the basis of the discrepancies between studies. Addressing these discrepancies is well beyond the scope of the current study.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Ex vivo hPCLS model
Use of our human PCLS for ex vivo studies was previously described. 95 Briefly, hPCLS were generated from lung tissues obtained from patients undergoing surgical operations at Hannover Medical School. Tissues used for PCLS generation that were obtained from lung tumor resections were confirmed as tumor-free by an experienced pathologist. The freshly obtained lung tissues were processed into circular slices that were 300 microns thick and 8 mm in diameter as previously described. 34 All donors provided informed consent as approved by the Hannover Medical School Ethics Committee (Ethics vote #8867_BO_K_2020). PCLS were maintained in DMEM/F12 medium (Gibco) supplemented with 2 mM of HEPES (Gibco), 13 GlutaMAX, 100 U/ml penicillin and 100 mg/mL streptomycin in a humidified 37 C and 5% CO 2 incubator.

Viruses
The pandemic H1N1 strain A/Netherlands/602/09 (NL09) and seasonal strain H3N2 A/Netherlands/241/11 (NL11) influenza viruses were obtained from the Repository of the National Influenza Center at the Erasmus Medical Center in Rotterdam, the Netherlands, and were grown on MDCKs for 48 h at 37 C. Virus stocks and culture supernatants were stored at À80 C until further use. Virus yields were titrated on MDCK cells by 50% tissue culture infectious dose (TCID 50 )/mL method as described by Reed and Muench. 96

METHOD DETAILS Inhibitors
Small molecule kinase inhibitors (SMKI) were all purchased from Selleckchem (TX, USA). Inhibitors were diluted in DMSO to a stock concentration of 10 mM and stored at À20 C upon usage.

In vitro cytotoxicity assays
In vitro cytotoxicity of SMKIs on mock-infected A549 cells was determined using CellTiter-Glo 2.0 (CTG) Cell Viability Assay (Promega (Sigma) and 50 ng/mL TPCK-treated trypsin (Sigma)). The cells were inoculated with the virus at the indicated multiplicity of infection (MOI) for 1h at 37 C. The cells were washed twice with PBS+/+ (Gibco) to remove unbound virus and incubated in infection medium at 37 C in the presence or absence of SMKIs at the indicated concentrations. Supernatants were collected at 0, 24, 48, 72 h post-infection (hpi), and viral titers were determined by TCID 50 assay in MDCK cells. 96 Prism 9.0 (GraphPad) Heatmap function was used for visualization. The assay's lower limit of detection (LoD) is 10 1 TCID 50 /mL, and its upper LoD is 10 9.5 TCID 50 /mL.

Immunofluorescent staining and imaging
To visualize virus infection, infected cells were fixed with 4% paraformaldehyde (4% PFA/PBS) (Roth) for 30 min at room temperature (RT), permeabilized with 0.1% Triton X-100 for 15 min at RT, washed with PBS and blocked with heat inactivated 5% horse serum (Sigma) in PBS (PBS-HS) at RT for 1h. Cells were then incubated with mouse monoclonal antibodies to IAV nucleoprotein (clone HB65, ATCC) diluted in PBS-HS at 0.2 mg/mL overnight at 4 C under constant agitation. Cells were washed and incubated with AlexaFluor-594 conjugated goat anti-mouse IgG antibody (0.2 mg/mL; ThermoScientific) and NucBlue Live ReadyProbes Reagent (ThermoScientific) for 1h at RT under constant agitation. Cells were washed 3 times with PBS (Gibco), images were captured using a Leica DMi8 fluorescence microscope and quantitative analysis was performed using ImageJ Threshold, Watershed, and Particle Analyser.
A previously described ImageJ Toolbox counting macro, 94 was used to quantify the number of nuclei and the number of separate infected cells by analyzing the RAW image data for each channel (n = 4). The nucleus count was used to define the total cell number per 0.6 mm 2 . The NP staining was used to define the number of infected cells per 0.6 mm 2 . The ratio of infected to total cells was used to calculate Relative Infectivity. The total number of cells based on nuclei detected relative to mock-infected cells treated with the respective NRTKI was used to determine Relative Viability. Prism 9.0 (GraphPad) Heatmap function was used for visualization.

Immunohistochemistry staining
Mock-and virus-Infected PCLS were inactivated by fixation in 4% PFA/PBS (Roth) and paraffin-embedded into blocks. Tissue sections (2 mm thick) were cut from the paraffin-embedded blocks and subjected to Hematoxylin & Eosin (HE) staining using standard protocols. Immunostaining for IAV antigen was done using an HRP-conjugated anti-IAV NP antibody. Histological analysis was performed by an experienced pathologist blinded to clinical data and experimental setup using a routine diagnostic light microscope (BX43, Olympus). Representative images were acquired with an Olympus CS50 camera using Olympus CellSens software (Olympus). Semi-quantitative analysis of IAV NP signal was performed for all tested NRTKIs using FIJI image-analysis software.
To measure polymerase activity during IAV infection, cells were infected at an MOI of 1 with NL09 or NL11 at 24h post-transfection (hpt) of the pPOLI-358-FFLuc reporter and the GFP plasmids in the presence or absence of SMKIs at the indicated concentrations as described above. At